1. Field of the Invention
The present invention relates to the disintegration of biologically active nucleic acids in a biologically active material by exposing the biological material, optionally treated with a photodynamic substance, to a laser beam which yields a non-infectious biological product having essentially all biologically active nucleic acid disintegrated while the biological integrity and activity, such as antigenicity, immunogenicity, protectivity or enzymatic characteristics, of the biological material is maintained. The invention relates also to the production of biologicals, such as vaccines, genetically engineered protein products, monoclonal antibodies or blood factors having essentially all biologically active nucleic acid disintegrated.
The use of continuous cell lines (CCL) for the production of therapeutic biological products and the problems of contaminating nucleic acids in biological products as well as the inactivation of residual contaminating cellular DNA in such biologicals have been discussed. While certain products, such as recombinant proteins, vaccines or monoclonal antibodies, are effectively produced using vertebrate cells, certain other protein products are not easily obtained from common used cells due to the low production rate of the proteins in those cells. Other mammalian cells which are easy to cultivate are not licensed by regulatory authorities for the production of genetically engineered proteins and for their use for therapeutic and prophylactic porposes in humans or animals.
It has been suggested to use mammalian cell lines for the genetically engineered manufacture of many proteinaceous products as well as for the production of virus vaccines. However, certain problems are anticipated in the use of such cell lines. Many products are not released or secreted from mammalian cells directly into the culture medium. Accordingly, the harvest of such products often will require rupturing cellular membranes to release those products into the medium from which they may subsequently be refined or purified. Such rupture, however, will also release cellular nucleic acid into the medium. Particularly because many easily cultured cell lines are transformed mammalian tumorogenic cell lines with an oncogenic potential, such as MDCK cells or SK-Hep cells, a need exists to ensure that active nucleic acid is not present as a contaminant in the proteinaceous end product or in the vaccine preparation. Attempts, therefore, have been made to inactivate cellular nucleic acids that might be associated with a risk of oncogenicity.
Vaccination against viral diseases has been one of the major tasks of medicine over the past century. While effective vaccines have been developed for a large number of diseases, development of safe and effective vaccines for a number of other diseases, e.g. HIV infections, remain problematic. The use of inactivated or killed viral agents as a vaccine, although generally safe, will not always be effective if the immunogenic characteristics of the agent are altered. However, if a highly infectious, virulent virus is used for vaccine manufacture, one has to be sure that the inactivation process results in a total loss of infectivity.
The use of live, attenuated viral agents as vaccines will often result in improved immunologic reactivity, but increases the risk associated with vaccination in that the vaccine itself is infectious. For example, the virus may revert to an active, virulent form and the organism may be able to propagate and disseminate leading to disease.
Thus, one must generally choose between improved effectiveness and greater degree of safety when selecting between live attenuated and inactivated vaccine preparations. The choice is particularly difficult when the virus is resistant to inactivation processes and requires highly rigorous inactivation conditions which are likely to negatively affect the antigenic characteristics.
Various techniques are known for killing or inactivating viruses for vaccine use. These include chemical or physical treatment e. g. inactivation with formaldehyde, hydroxylamine, .beta.-propiolactone, UV irradiation, or photodynamic dyes, e.g. methylene blue and visible light. Despite these general disclosures, a need exists to improve the selectivity of such inactivation methods such that protein viral coats will better retain their integrities while inactivation/kill rates are enhanced.
One major problem of inactivated virus vaccines is that if the inactivation process of the viral nucleic acid is not complete, there is the risk that virus nucleic acid may be administered to a host associated with the virus vaccine, and under certain circumstances this may itself be infectious. E.g. HIV RNA could be reverse transcribed by viral or cellular enzymes and be integrated into the host genome, leading to HIV replication.
Many attempts have been made to inactivate viruses in a sample, such as vaccines or plasma, or cellular nucleic acids while preserving the antigenic and immunogenic properties of the proteins in that sample.
2. Description of Related Art
U.S. Pat. No. 5,106,619 discloses a viral vaccine with preserved antigenic and immunogenic characteristics produced by inactivation of enveloped and non-enveloped viruses with UV light in the presence of fourocoumarin in a non-oxidizing atmosphere.
U.S. Pat. No. 4,880,512 discloses a method for treating biological media such as blood fractions, genetically engineered protein products and vaccine preparations and the photolysis of nucleic acids in the presence of proteins, while preserving the antigenic and immunogenic properties of the protein. Biological media comprising tryptophan-containing proteins were irradiated with pulsed light of different wavelengths and flux.
Matthews et al. (1992, Blood Cells 18:75-89) reported about inactivation of viruses in virus-infected cells in culture media or in whole blood by irradiation with filtered xenon light source and/or a tuneable dye laser in the presence of a photodynamic dye. Prodouz et al. (1987, Blood 70:589-592) disclosed a method for virus inactivation in samples by UV irradiation using a radiation at 308 nm of XeX1 eximer laser. In WO 96/00091 samples are irradiated with electro-magnetic radiation generated by a tuneable laser-device at a wavelength in the range of from 300 to 370 nm for virus inactivation.
Perrin et al. (1995, Biologicals 23:207-211) used .beta.-propionlactone for the inactivation of residual contaminating cell DNA in biologicals. Although this agent is known to inactivate viruses, its use is difficult to control because of the lability and reactivity of the agent. It rapidly hydrolyses in aqueous solutions and it can also modify the product e.g. by reducing Fc function in immunoglobulins.
The use of nucleases to inactivate DNA or RNA has also been suggested, but it is known that this is not a very effective procedure in that a high percentage of nucleic acid may be associated with protein and thus be inaccessible to nucleases.
It is known that photosensitive dyes and, in particular phenothiazine dyes, such as methylene blue, bengal rose, neutral red or toluidine blue, in the presence of light or UV light can inactivate viruses. This is caused by the preferred affinity of the photodyes to nucleic acids.
The reactivity of phenothiazine dye with viruses has been studied since 1930. Perdrau et al. (1933, Proc. Roy. Soc. 122:288-298) reported that a wide range of viruses can be inactivated by the action of methylene blue in the presence of light. Rabiesvirus, influenza virus, Juninvirus, canine distemper virus, HIV and herpes simplex virus were shown to be inactivated by methylene blue and light irradiation (Swartz et al., 1979, Proc. Soc. Exp. Biol. Med., 161:204-209; Schnipper et al., 1980, J. Clin. Invest., 65:432-438; Cobo et al., 1986, Med. Microbiol. Immunol., 175:67-69, Bachmann et al.,1995, J. Med.Virol. 47:172-178). Enveloped viruses, in general, possess some inherent photosensitivity, while non-enveloped viruses are photoresistant (Wallis et al., 1964, Virology 23:520-527).
EP 0 196 515 discloses a method for inactivation of viruses in a therapeutic protein composition by exposing the composition to light in the presence of a photosensitizer. Preferred photosensitizers used were protoporphyrin and chlorpromazine. It was found that viruses could be inactivated, but that under the conditions tested some inactivation of Factor VIII also occurs.
U.S. Pat. No. 4,181,128 discloses a system for inactivation of microorganisms, such as viruses, bacteria, toxins and tumor cells by treatment with methylene blue, light, electricity and an oxgygen atmosphere.
U.S. Pat. No. 4,950,665 discloses a method for using methylene blue to selectively derivatize guanosine and to inactivate virus and cancerous cells in vivo.
Treatment of plasma with methylene blue and light leads to inactivation of enveloped viruses including HIV, HSV, VSV and some non-enveloped viruses such as SV40 (Mohr et al., 1992, Ann. Hematol. 65:224-228). Although it is believed that nucleic acids are the preferred targets of methylene blue/light treatment (Tuite et al., 1993, Photochem. Photobiol. B: Biol. 21:103-124), some alterations in capsid proteins of methylene blue-photoinactivated viruses as well as a loss of the activity of plasma proteins has been observed (Specht et al., 1994, Photochem. Photobiol. 59:506-514; Bachmann et al., 1995, J. Med. Virol. 47:172-178, Mohr et al., 1995, Immunol. Invest. 24:73-85).